Alphaviruses are currently being used as vector platforms to develop vaccines for infectious diseases and cancer (e.g., see U.S. Pat. Nos. 5,792,462; 6,156,558; 5,811,407; 6,531,135; 6,541,010; 6,783,939; 6,844,188; 6,982,087; 7,045,335; 5,789,245; 6,015,694; 5,739,026; Pushko et al., Virology 239(2):389-401 (1997), Frolov et al., J. Virol. 71(1):248-258 (1997); Smerdou and Liljestrom, J. Virol. 73(2):1092-1098 (1999)). Alphaviruses comprise a genus in the Togaviridae family, and members of the genus are found throughout the world, in both vertebrate and invertebrate hosts. Among the most studied alphaviruses for vector platforms are Venezuelan Equine Encephalitis (VEE) Virus, Semliki Forest Virus (SFV), and Sindbis Virus (SV), the prototype member of the genus.
One such vector platform is the alphavirus replicon system, described in U.S. Pat. No. 6,190,666 to Garoff et al., U.S. Pat. Nos. 5,792,462 and 6,156,558 to Johnston et al., U.S. Pat. Nos. 5,814,482, 5,843,723, 5,789,245, 6,015,694, 6,105,686 and 6,376,236 to Dubensky et al; U.S. Published Application No. 2002-0015945 A1 (Polo et al.), U.S. Published Application No. 2001-0016199 (Johnston et al.), Frolov et al. (1996) Proc. Natl. Acad. Sci. USA 93:11371-11377 and Pushko et al. (1997) Virology 239:389-401. An alphavirus replicon vector is engineered to contain and express one or more nucleic acids of interest, where the nucleic acid of interest can encode, for example, an antigen, a cytokine, a ribozyme, or an enzyme. The alphavirus replicon vector can be derived from any alphavirus, such as Venezuelan Equine Encephalitis (VEE) virus, Sindbis virus, e.g., strain TR339, South African Arbovirus No. 86, and Semliki Forest virus, among others. The vector is then introduced into cells in culture that allow replication of alphaviruses and in which the structural proteins of the alphavirus are also expressed, so that the vector is packaged by the alphavirus structural proteins into alphavirus replicon particles (ARPs). ARPs are then harvested from the culture and delivered into subjects for a variety of therapeutic purposes.
Various constructs have been developed to enhance immunogenicity and effectiveness of the ARP system in vaccine applications. Many of these constructs have also been designed to decrease the likelihood of formation of replication-competent alphavirus through recombination of genome fragments. Johnston et al. (U.S. Pat. Nos. 5,792,462 and 6,156,558) recognized the potential for recombination from a single helper system (in which the complete set of structural protein genes of an alphavirus are on one RNA molecule and the nonstructural protein genes and heterologous nucleic acid of interest are on a separate replicon RNA), and thus designed “double-helper” systems that utilized two helper RNAs to encode the structural proteins. Dubensky et al. (U.S. Pat. No. 5,789,245) and Polo et al. (U.S. Pat. No. 6,242,259) describe the use of two DNA alphavirus structural protein expression cassettes, stably transformed into a packaging cell line, to package alphavirus vectors by production of RNAs expressing those structural proteins upon introduction of a replicating alphavirus vector into cultures of the packaging cell. Liljestrom and colleagues have presented data confirming that a “single helper system” will generate wild-type alphavirus particles (Berglund, et al. Biotechnology 11(8): 916-920 (1993)). Smith et al have described other novel RNA helpers that direct expression of the structural proteins (WO 2004/085660).
By distributing the viral coding sequences among three nucleic acids, two of which comprise the helper system, as described above, the theoretical frequency of recombination that would create a replication-competent virus (“RCV”) is reduced significantly relative to single helper systems. These systems include the use of the alphaviral subgenomic promoter, often referred to as the 26S promoter or the viral junction region promoter, to provide a construct which functions as an independent transcriptional unit and the use of the alphavirus RNA polymerase recognition signals, so that the helper systems can take advantage of the presence of the alphavirus replication machinery for amplification and efficient expression of helper functions.
In existing systems, known packaging signals are typically included in replicon RNAs and excluded from helper constructs. However, helper RNAs are nonetheless packaged or copackaged at a lower frequency (Lu and Silver. J. Virol Methods, 91(1):59-65 (2001)), and helper constructs with terminal recognition signals will be amplified and expressed in the presence of a replicon, potentially yielding recombination events with other helper molecules or the replicon RNA.
Animal studies with alphavirus replicon particles have employed doses ranging from 105 to 108, with 107, 5×107, and 108 having been effectively employed in non-human primates, which are also the doses being used in human clinical trials. In addition, higher doses such as 2×108, 5×108 and 109 are also useful in applications for humans. Such dosages require large scale manufacturing procedures, and at such scale, it is statistically possible that replication-competent alphavirus may be generated with existing RNA helper systems.
Thus, there remains a need in the art to provide improved systems for manufacturing alphavirus replicon particles to further reduce the predicted frequency for formation of replication-competent alphavirus, and to optimize manufacturing strategies and costs.
The present invention provides alphavirus RNA helper molecules encoding alphavirus structural proteins that lack a promoter sequence, thereby significantly decreasing the theoretical number of functional recombination events that might occur between the helper molecules and the replicon vector, resulting in a decrease in the theoretical prediction for the rate of formation of replication-competent alphavirus during the production of recombinant alphavirus particles.